EP2406377B1 - Séquence polynucléotidique et polypeptidique et ses procédés - Google Patents

Séquence polynucléotidique et polypeptidique et ses procédés Download PDF

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EP2406377B1
EP2406377B1 EP10750465.6A EP10750465A EP2406377B1 EP 2406377 B1 EP2406377 B1 EP 2406377B1 EP 10750465 A EP10750465 A EP 10750465A EP 2406377 B1 EP2406377 B1 EP 2406377B1
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seq
precursor
vector
set forth
insulin
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EP2406377A2 (fr
EP2406377A4 (fr
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Mulakkapurath Narayanan Manoj
Venkata Ramachandra Rao Vasamsetty
Madhuri Baliga
Kirubakaran Naveen Kumar
Chandrasekhar Bhaskaran Nair
Pillarisetti Venkata Subbarao
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Bigtec Pvt Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present disclosure relates to a field of recombinant DNA therapeutics. It involves the bio-informatics design, synthesis of artificial gene for human insulin precursor including leader peptide coding sequence, cloning in an expression vector and expression in an organism, preferably Pichia pastoris.
  • the present disclosure also relates to methods of downstream processing for obtaining protein precursor molecules and subsequent conversion of precursor molecules to functional proteins.
  • Human Insulin is a polypeptide hormone involved in regulation of glucose in blood and body fluids. Production deficiency leads to type 1 or type 2 diabetes. Type 1 is especially Insulin dependent diabetes. Earlier, Insulin used to be supplemented from animal sources (bovine and pig), which often results in undesirable allergic immune response or hypersensitive reaction on continual administration for longer periods. The next generation of Humanized Insulin was produced in E. coli by recombinant DNA technology and is being successfully used for the past several years. Although recombinant human Insulin is being expressed in different hosts through patented processes to meet the diabetes therapeutic requirements, the demand is growing and forcing the man kind to explore new and modified methods to produce commercially viable quantities.
  • Recombinant human Insulin currently available in the market is produced from at least three different expression systems i.e. E. coli, Pichia pastoris and Hansenula polymorpha. Over expression in E. coli results in proteins accumulating as insoluble inclusion bodies. Solubilization and refolding of the recombinant Insulin from the inclusion bodies requires use of chaotropic chemicals such as guanidine hydrochloride, urea, etc. and presence of traces of these chemicals in the final product even after extensive purification could be hazardous. Alternatively, proteins can be expressed in yeast system and secreted out into the medium at much higher levels in soluble form. However, levels of expression obtained in each yeast system differed from protein to protein for unknown reasons.
  • the two chains of Human Insulin are also being expressed separately using two different vectors and assembled together in-vitro after purification. Disulphide linkages between two chains is facilitated by chemical methods.
  • WO 99/37793 describes a process for making desired polypeptides in yeast. Desired products are expressed as leader bound polypeptides comprising a-factor leader peptide and have a monobasic (Lys or Arg) processing site.
  • the present invention is defined by the appended claims and is therefore directed to a polynucleotide sequence, a method for obtaining a recombinant insulin precursor molecule, a method for obtaining a recombinant insulin molecule, a recombinant vector and a recombinant host cell as defined in those appended claims.
  • the present disclosure relates to a polynucleotide sequence as set forth in SEQ ID NO: 2; a polypeptide sequence as set forth in SEQ ID NO: 1.; a method for obtaining recombinant insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1, said method comprising steps of: a) synthesizing a polynucleotide sequence set forth in SEQ ID NO: 2 by combining 26 oligonucleotides of SEQ ID NOS: 3 to 28 by assembly PCR, and inserting the synthesized sequence in a vector, b) transforming a host cell with said vector followed by antibiotic screening host selection, and c) fermenting the selected transformed host cell and in-situ capturing of the insulin precursor molecule to obtain said precursor having polypeptide sequence as set forth in SEQ ID NO: 1; a method of downstream processing for in-situ capturing of protein precursor molecule during fermentation process, said method comprising steps of: a) simultaneous pumping of fermentation product obtained during fermentation into
  • the present disclosure relates to a polynucleotide sequence as set forth in SEQ ID NO: 2.
  • the polynucleotide encodes a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
  • the present disclosure relates to a polypeptide sequence as set forth in SEQ ID NO: 1.
  • the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
  • polypeptide sequence corresponds to polynucleotide sequence set forth in SEQ ID NO: 2, wherein the polynucleotide is subjected to post-transcriptional modification and codon optimization to obtain corresponding polypeptide of SEQ ID NO: 1.
  • the present disclosure relates to a method for obtaining recombinant insulin precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1, said method comprising steps of:
  • the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
  • the synthesized polynucleotide and the vector are subjected to restriction enzyme digestion for insertion of the polynucleotide into the expression vector and wherein restriction enzymes are selected from a group comprising BamHI, NotI, SacI, BgIII and SacII or any combination thereof.
  • the vector is selected from a group comprising pPIC9K and pPICZ ⁇ , preferably pPIC9K and wherein the host is selected from a group comprising Pichia pastoris, Pichia methanolica; Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris.
  • the in-situ capturing of the precursor molecule is carried out by hollow fibre harvesting system and ion-exchange chromatographic column to obtain said precursor.
  • the present disclosure relates to a method of downstream processing for in-situ capturing of protein precursor molecule during fermentation process, said method comprising steps of:
  • the permeate comprise of clarified cell free broth and the retentate comprise of concentrated cells.
  • the wherein the retentate is recycled back to fermentor vessel along with fresh medium in the fermentor and the permeate is passed through the ion-exchange chromatographic column for capturing the protein precursor.
  • the protein precursor selectively binds to polymer matrix of the ion-exchange chromatographic column and is eluted with the elution buffer.
  • the present disclosure relates to a method of downstream processing for in-situ conversion of protein precursor molecule into functional protein molecule, said method comprising step of:
  • the precursor molecule is concentrated to a range of about 100 mg/ml to about 200 mg/ml.
  • the organic solution comprise of O-tert -Butyl-L-theronine tert-butyl ester acetate dissolved in 1:1 v/v dimethyl sulfoxide (DMSO) : Methanol and the deblocking buffer comprise a combination of tryptophan and trifluoroacetic acid.
  • DMSO dimethyl sulfoxide
  • the TPCK column is equilibrated with a combination of CaCl 2 and Acetic acid and the hydrophobic interaction chromatographic column is equilibrated with a combination of Acetic acid and Ammonium sulphate.
  • time for the incubation ranges from about 1.5 hrs to about 3.5 hrs and temperature for the incubation ranges from about 15°C to about 25°C.
  • the present disclosure relates to a method for obtaining recombinant insulin molecule from a precursor molecule having polypeptide sequence as set forth in SEQ ID NO: 1, said method comprising steps of:
  • the polypeptide is a fusion polypeptide comprising recombinant Human Insulin Precursor and signal peptide.
  • the synthesized polynucleotide and the vector are subjected to restriction enzyme digestion for insertion of the polynucleotide into the expression vector and wherein the restriction enzymes are selected from a group comprising BamHI, NotI, SacI, BgIII and SacII or any combination thereof.
  • the vector is selected from a group comprising pPIC9K and pPICZ ⁇ , preferably pPIC9K and wherein the host is selected from a group comprising Pichia pastoris, Pichia methanolica, Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris.
  • the in-situ capturing of the precursor molecule is carried out by hollow fibre harvesting system and ion-exchange chromatographic column to obtain said precursor.
  • the in-situ conversion of the precursor molecule is carried out by subjecting the precursor molecule to TFF Cassette and TPCK trypsin immobilized column to obtain protein ester.
  • the protein ester is subjected to deblocking buffer followed by hydrophobic interaction chromatographic column to obtain said recombinant insulin molecule.
  • the present disclosure relates to a recombinant vector comprising polynucleotide sequence set forth in SEQ ID NO: 2.
  • the vector is selected from a group comprising pPIC9K and pPICZ ⁇ , preferably pPIC9K.
  • the present disclosure relates to a recombinant host cell, transformed by introduction of a vector comprising polynucleotide sequence set forth in SEQ ID NO: 2.
  • the host is selected from a group comprising Pichia pastoris, Pichia methanolica, Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris and wherein the vector is selected from a group comprising pPIC9K and pPICZ ⁇ , preferably pPIC9K.
  • the main object of the present disclosure is to de novo design and express the gene coding for "secretion signal and recombinant Insulin Precursor fusion protein" comprising the amino acid sequence as set forth in SEQ ID NO: 1.
  • the present disclosure relates to a method for obtaining recombinant human insulin, said method comprising the steps of:
  • optimization of nucleotide sequence was done for enhanced expression and secretion of target protein into fermentation medium.
  • said SEQ ID No. 2 is obtained by multiple stages in-silico optimization of nucleotide sequence based on "Codon-Pair Frequency" of highly expressed proteins in Pichia pastoris.
  • the sequence was further tuned to enhance protein synthesis by mRNA secondary structure prediction and removing high melting stem loop structures, which enables un-restricted ribosome movement and high speed protein synthesis.
  • Codon optimisation is a method of gene optimisation, where in the synthetic gene sequence is modified to match the "codon usage pattern" for a particular organism.
  • select "most frequently used codons” from a list of degenarate codons for an aminoacid), by that organism. So that the aminoacid sequence remains same but with a different DNA sequence, matched for that organism. How-ever this does not consider the fact that codons are read by ribosomes in "pairs", during protein synthesis. There are 2 codon binding site in ribosome, on adjescent places. Extensive analysis was done and a particular pattern was observed in which the "codon-pairs" are used by pichia pastoris.
  • the whole gene sequence i.e. Insulin Precursor and secretion signal was subjected to optimization together, as it is expressed as a single chain protein in the expression host.
  • said precursor is constructed with about 26 oligonucleotides coding for Insulin precursor- Signal peptide fusion protein.
  • said vector is selected from a group comprising pPIC9K and pPICZ ⁇ , preferably pPIC9K
  • said cloning is carried out at downstream of AOX1 promoter in pPIC9K vector.
  • said host is selected from a group comprising Pichia pastoris, Pichia methanolica, Pichia guilliermondii and Pichia caribbica, preferably Pichia pastoris.
  • said cloning was carried out by simultaneous multiple gene insertions and direct selection using an antibiotic to get high copy number of gene into the host
  • said fermentation is carried out in a modified low salt minimal medium at optimal temperature range, aeration, cell densities and feeding, which enables high level expression and easy downstream processing.
  • fermentation process and harvesting process are coupled. It involves a hollow fibre harvesting module is connected to fermenter for in-situ filtration of broth during harvesting.
  • the culture from fermenter is pumped to a hollow fibre cassette to separate cell free broth from the cells.
  • the cells after filtration are recycled back to fermenter vessel along with medium to maintain culture volume and promote normal growth of culture.
  • capturing of human insulin precursor is coupled with trypsin digestion and transpeptidation in an immobilized trypsin column. It involves binding of insulin precursor in cell free broth from hollow fibre filtration system to a chromatography column packed with high binding capacity synthetic resin. The unbound is again channeled back into fermenter along with fresh medium.
  • the bound protein is eluted and further channeled into a column packed with TPCK trypsin immobilized to matrix.
  • the eluted precursor is mixed with necessary buffers and desired PH.
  • the precursor is converted into insulin ester by tryptic digestion and transpeptidation in the column.
  • the insulin ester is eluted from column and deblocked to convert into human insulin and lyophilized.
  • the human insulin is polished to highest purity by reverse phase chromatography.
  • said fermentation medium has a pH ranging from about 4.0 - 5.0, preferably about 4.75 during initial phase of fermentation; about 4.0 - 5.0, preferably about 4.80 during glycerol phase and about 4.0 - 5.0, preferably about 4.95 during induction phase.
  • said temperature at fermentation ranges from about 29 - 30°C, preferably about 30.0°C for batch phase; about 29 - 30 °C, preferably about 29.5 °C for glycerol fed batch; and about 27 - 29 °C, preferably about 28.0 °C for induction phase with methanol.
  • said aeration at fermentation ranges from about 0.5 - 1.5 VVM pure air, preferably 1VVM pure air for batch phase; about 0.5 - 1.5 VVM air: oxygen, preferably about 1.0 VVM air: oxygen (about 90:10) for glycerol batch; and about 1.5 VVM air: oxygen ratio begins at about 85:15 and ends at about 40:60 with an increment/decrement of about 5 at about every 5 hours for methanol batch (induction phase).
  • glycerol feeding is carried out to promote high cell density growth before induction and is continued until cell density (OD 600 ) reaches about 500. Then methanol is fed exponentially to promote increased expression of target protein.
  • a synthetic gene having modified nucleotide sequences and coding for a gene comprising the Mat- ⁇ secretion signal, spacer, and the insulin precursor was designed de novo. Extensive bioinformatics analysis was used to arrive at a novel coding sequence, based on nucleotide patterns from highly expressed proteins in Pichia pastoris.
  • the synthetic gene (482bp) was constructed by synthesizing 26 oligonucleotides and combining them by assembly PCR.
  • Pichia expression system is known for its very high levels of expression, using a methanol inducible promoter. Proteins can be expressed as secretory proteins and therefore purification of the same becomes simple. The doubling time of the strain, ease of handling, minimal growth requirements, availability of convenient vectors, host systems and selection methodologies make Pichia pastoris an ideal and attractive system for study. High cell densities are achievable in minimal mineral media and the ease of induced expression of proteins adds to the convenience of using this system for recombinant protein expression.
  • the insulin precursor fusion protein gene obtained by assembly PCR was confirmed by DNA sequencing ( Fig. 05 ) and was cloned into Pichia pastoris expression vector pPIC9K.
  • the vector after linearization transformed into GS 115 strain of Pichia by electroporation.
  • the expression cassette was integrated into the Pichia host system by homologous recombination. Clones harboring high copy number inserts were picked by antibiotic screening. Clones showing maximum resistance to the antibiotic genticin (G418) were picked and screened for their ability to express and secrete the Insulin precursor into the culture medium. Promising clones were further evaluated by 7 liter capacity fermenter. The fermentation yield of insulin precursor is around 1.5 gm/litre. This can be further increased through additional optimization of the process.
  • the secreted insulin precursor was captured from the broth, purified and enzymatically modified to obtain Human Insulin.
  • Biological activity of the final product in terms of regulating blood glucose has been established in mice and rats and found to be comparable with commercially available therapeutic recombinant Human Insulin formulations.
  • the process has been optimized at multiple steps, which has cumulative effects and resulted in increased yields.
  • the "codon-pair sequence" of the entire coding region stability of mRNA, multiple copy insertions, optimized media components and growth and induction parameters.
  • fermentation yields are high.
  • Oligonucleotides [as given in SEQ 3] coding for the fusion protein "Mat- ⁇ - Insulin Precursor" fusion protein were designed and custom synthesized. These oligonucleotides were assembled by assembly PCR. The PCR product is double digested with restriction enzymes BamHI and NotI ( Fig. 04 ) and ligated into similarly processed vector pPIC9K ( Fig. 03 ) using T4 DNA ligase.
  • Table 1 Reaction Mix; using Phusion High Fidelity DNA Polymerase (NEB) Kit PCR MIX (50 ⁇ l) 5X rxn buffer (Hi Fidelity) 10.0 ⁇ l 10mM dNTPs 1.0 ⁇ l Oligos 26 ⁇ l Taq 0.5 ⁇ l (1 unit/ ⁇ l) MilliQ 12.5 ⁇ l Total volume 50 ⁇ l
  • Table 2 PCR Program for 1st Assembly PCR PCR MIX (50 ⁇ l) Step 1 Initial denaturation 98°C 30 sec 1 cycle Step 2 Denaturation 98°C 10 sec Step 3 Annealing 57°C 30 sec 30 cycles Step 4 Extension 72°C 30 sec Repeat 2,3 & 4 30 times Step 5 Final extension 72°C 7 min 1 cycle Step 6 Final Hold 4°C ⁇
  • the product of assembly PCR is used as template for 2 nd PCR. Product quantity is not increased in exponential way, hence it is not checked on gel electrophoresis.
  • the product from assembly PCR is directly used as template for second PCR where the assembled gene is amplified by using AOX1 primers.
  • Amplicon obtained from second PCR and size of the amplicon is matching with the size ⁇ 500 bp (482 bp).
  • the product of second PCR is extracted from agarose gel for sequencing
  • the ligation mix i.e. pPIC9K vector containing the ligated gene of interest (insulin precursor+ mat- ⁇ secretion signal) is used for transformation into chemically competent TOP 10 E . coli strain ( Fig. 01 ).
  • CaCl 2 was used for competent cell preparation. Transformation was done by heat shock method. Transformation mix was plated on LB medium containing Ampicillin in order to select transformed colonies. Colonies were obtained after incubation of plates at 37°C for 12-14 h. Glycerol stocks of transformed cells were prepared and stored at -70°C. Plasmid from E.coli is prepared by the protocol from Promega Kit (Wizard plus SV minipreps DNA purification system).
  • Recombinant pPIC9K plasmid vector is linearized with restriction enzymes SacI/BglII/salI, purified, quantified and used for transformation into Pichia pastoris. Approximately 10 ⁇ g of the linearized plasmid DNA with insert were used for electroporation of electrocompetent host cells. The specifications used for electroporation are 760 Volts/ 5 milli seconds in 2 mm cuvette.
  • the transformation mixture was incubated in 1M sorbitol for 30 min for cells to recover and further incubated in liquid regeneration media for 4 hours at 30°C with shaking. Cells were then plated on to minimal media lacking histidine and containing antibiotic G418. The His + colonies that grew on these plates were screened by PCR by using AOX1 primers ( Fig. 02 ) PCR positive cell lines are plated on fresh RD medium plates for further screening of high copy number lines.
  • Clones containing multiple copies of the gene inserted into the genome were further screened using higher concentrations of antibiotic G418. Colonies resistant to more than 4mg G418 are considered to contain more than twelve copies of the gene. Such colonies were selected, grown on YPD medium and maintained as glycerol stock at - 70°C.
  • Each colony to be screened was grown in 5 ml YPD in a culture tube by incubating at 30°C / 230rpm / 24hrs.
  • the seed (1ml) is inoculated to 50 ml BMG (buffered minimum glycerol medium) in 250ml Erlenmeyer flask and incubated at 30°C / 220rpm / 24hrs.
  • Cells were harvested by centrifuging at 2000g/ 5 minutes at room temperature. Supernatant was decanted and the cell pellet was resuspended in 25ml BMM (buffered minimum methanol medium) in 150ml baffled flasks and then allowed to grow at 30°C / 200rpm for 3days.
  • the culture was induced with methanol to a final concentration of 1.0% at every 24hrs. Samples were taken at 24 hr intervals and analyzed by HPLC and SDS-PAGE.
  • Colonies showing good expression were made into glycerol stocks for further evaluation at fermentation level.
  • Fermentation was carried in in-situ autoclavable automated vessel (BioFlo 415, NBS) of 7 litre capacity. All parameters like agitation, gas flow rates, feeding, pH adjustments, antifoam were controlled by PID controller.
  • glycerol stock is inoculated and grown in YPD medium by shake flask culture for 18 - 20 hrs at 220 rpm/30°C till OD 600 reaches 10-12.
  • the first seed is again inoculated onto YPG medium and grown at above mentioned conditions.
  • culture reached log phase around 20 hrs
  • OD 600 around 25 -30 the cells are harvested at 1500g/5min and suspended in autoclaved milliQ water. Then cells are inoculated into basal salt medium in fermenter upto OD 600 of 5.0.
  • Batch phase The fermenter medium pH adjusted to 4.75 before inoculation to avoid precipitation of medium if any.
  • Dissolved oxygen (DO) probe is also calibrated before inoculation. Trace metal solution of 8% added to the vessel before and after inoculation at fixed intervals. Temperature of the culture is maintained at 30°C. Vessel aeration was maintained as 1.0 VVM pure air. Initial batch phase last for 18 hrs until OD 600 reaches 120-150 with an indication of DO shoot up.
  • DO Dissolved oxygen
  • Glycerol fed batch The glycerol fed batch started with feeding of 50% glycerol containing 12% trace metal solution on exponential feed rate to achieve high cell density before induction. Temperature and pH were maintained at 29.5°C and 4.80. Vessel aeration was maintained as 1.0 VVM air and oxygen in a ratio of 9:1.
  • Methanol batch Induction of Insulin Precursor (IP) was started by feeding 100% methanol containing 12% PTM4 trace metal solution. Initial methanol feed was given as spikes until culture gets adapted, subsequently switched on to exponential feed. The DO spike method was used to determine ramp of methanol feed. Methanol feed for Mut + and Mut s clones were based on Stratton et al., (Pichia protocols, Methods in Molecular Biology, Vol.103 ). Residual methanol in the vessel is continuously monitored using an in-house designed methanol probe and sensor connected to the vessel. Consumption of methanol signals increase in vessel temperature which is maintained at 28.5°C through out methanol fed batch. Medium pH was maintained at 4.95.
  • Vessel aeration was maintained as 1.5 VVM due to high density with air and oxygen in a ratio begins at 85:15 and ends at 40:60 with an increment/decrement of 5 at every 5 hrs.
  • induction phase samples were analyzed at 6-hours interval to check growth, induction and contamination if any.
  • Induced protein secreted into broth is analyzed by HPLC using 0.1% TFA / Acetonitrile solvents in C18 column. HPLC samples at 6 hour intervals showed progressive increase in protein level ( Fig. 07 ).
  • Fermentation samples were also analyzed by electrophoresis (SDS-PAGE) to assess the expression of insulin precursor and its increase with induction time ( Fig. 06 ).
  • Fermentation samples during induction phase are periodically checked to know any protease activity by azocasein assay. Fermentation conditions were optimized for high level expression of insulin precursor which is more than 65% of total proteins present in the final sample. Results showed that the total protein present in the final sample is ranging from 2.3 g/L with insulin precursor being 1.5g/L.
  • the induced culture When the induced culture is more than 36 hrs old, it is pumped from fermenter into hollow fiber harvesting system with 0.2 ⁇ cartridge via a peristaltic pump.
  • the permeate contains clarified cell free broth and retentate contains concentrated cells.
  • the retentate with cells is recycled back to fermenter vessel along with fresh medium to maintain normal growth and volume ( Fig. 11 ).
  • the permeate containing clarified cell free broth is passed through column packed with strong cation exchanger resin, SP sepharose (methacrylic polymer with sulphopropyl functional derivatization - GigaCap S 650, Toyopearl) at pH 3.0. for protein capturing.
  • SP sepharose methacrylic polymer with sulphopropyl functional derivatization - GigaCap S 650, Toyopearl
  • the column after protein binding is washed with 2 column volumes of pH 3.0 Tris buffer.
  • the Insulin precursor selectively binds to the polymer matrix and is eluted with Tris buffer at pH 8.0.
  • the chromatographic purity of insulin precursor is around 75% as checked on HPLC ( Fig. 08 ) and step yield is around 90 % w/w.
  • the PIP obtained in Example 4 is converted to Human Insulin via trypsin mediated digestion and transpeptidation followed by deblocking.
  • Insulin precursor eluted from ion exchange column is passed through 1kda MWCO TFF cassette and concentrated to 100-200mg/ml and its pH is adjusted to 7.3 with 1 N HCl.
  • the concentrated PIP is mixed with O-tert -Butyl-L-theronine tert-butyl ester acetate dissolved in 1:1 v/v dimethyl sulfoxide (DMSO) : Methanol.
  • DMSO dimethyl sulfoxide
  • reaction mixture is passed through TPCK-treated trypsin immobilized column (25ml XK column with cooling jacket) equilibrated with 50 mM CaCl 2 and 0.5 % acetic acid.
  • TPCK-treated trypsin immobilized column 25ml XK column with cooling jacket
  • reaction mixture is completely loaded into the column, the column is closed for 2 - 3 hrs to permit incubation of reaction contents and column temperature is maintained at 20°C.
  • the insulin precursor converted to Insulin butyl ester is eluted and checked by HPLC ( Fig. 09 ).
  • the purified Human Insulin meets the quality norms as per monograph of recombinant Human Insulin under British Pharmacopoeia 2007, by HPLC analysis ( Fig. 10 ) SDS PAGE (15%) ( Fig. 13 ) and western blot ( Fig. 14 ) of purified human insulin and comparison with commercial formulation is provided and.

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Claims (8)

  1. Séquence polynucléotidique telle que décrite dans SEQ ID NO:2, où le polynucléotide code pour un polypeptide de fusion comprenant un précurseur de l'insuline humaine recombinant et un peptide signal.
  2. Procédé pour obtenir une molécule précurseur de l'insuline recombinante possédant la séquence polypeptidique telle que décrite dans SEQ ID NO: 1, ledit procédé comprenant les étapes consistant à :
    a) synthétiser une séquence polynucléotidique décrite dans SEQ ID NO: 2 en combinant 26 oligonucléotides de SEQ ID NO: 3 à 28 par une PCR d'assemblage, et insérer la séquence synthétisée dans un vecteur,
    b) transformer une cellule hôte avec ledit vecteur suivi d'une sélection de l'hôte par un criblage avec un antibiotique, et
    c) fermenter la cellule hôte transformée sélectionnée et capturer in situ la molécule précurseur de l'insuline afin d'obtenir ledit précurseur possédant la séquence polypeptidique telle que décrite dans SEQ ID NO: 1.
  3. Procédé pour obtenir une molécule d'insuline recombinante à partir d'une molécule précurseur possédant la séquence polypeptidique telle que décrite dans SEQ ID NO: 1, ledit procédé comprenant les étapes consistant à :
    a) synthétiser une séquence polynucléotidique décrite dans SEQ ID NO: 2 en combinant 26 oligonucléotides de SEQ ID NO: 3 à 28 par une PCR d'assemblage,
    b) insérer la séquence synthétisée dans un vecteur et transformer une cellule hôte avec ledit vecteur suivi d'une sélection de l'hôte par un criblage avec un antibiotique,
    c) fermenter la cellule hôte transformée sélectionnée suivi d'un traitement en aval pour la capture in situ de la molécule précurseur de l'insuline, et
    d) convertir in situ la molécule précurseur de l'insuline possédant la séquence polypeptidique telle que décrite dans SEQ ID NO: 1 en ladite molécule d'insuline recombinante.
  4. Procédé selon la revendication 2 ou 3, dans lequel la capture in situ de la molécule précurseur est réalisée par un système de récolte à fibres creuses et une colonne de chromatographie échangeuse d'ions afin d'obtenir ledit précurseur.
  5. Procédé selon la revendication 3, dans lequel la conversion in situ de la molécule précurseur est réalisée en soumettant la molécule précurseur à une cassette TFF et à une colonne de trypsine TPCK immobilisée afin d'obtenir un ester de protéine.
  6. Vecteur recombinant comprenant la séquence polynucléotidique décrite dans SEQ ID NO: 2.
  7. Cellule hôte recombinante, transformée par l'introduction du vecteur selon la revendication 6.
  8. Cellule hôte selon la revendication 7, où l'hôte est sélectionné dans un groupe comprenant Pichia pastoris, Pichia methanolica, Pichia guilliermondii et Pichia caribbica, de préférence Pichia pastoris et où le vecteur est sélectionné dans un groupe comprenant pPIC9K et pPICZa, de préférence pPIC9K.
EP10750465.6A 2009-03-12 2010-03-12 Séquence polynucléotidique et polypeptidique et ses procédés Not-in-force EP2406377B1 (fr)

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US9223951B2 (en) 2014-02-07 2015-12-29 Bank Of America Corporation User authentication based on other applications
US9208301B2 (en) 2014-02-07 2015-12-08 Bank Of America Corporation Determining user authentication requirements based on the current location of the user in comparison to the users's normal boundary of location
US9721268B2 (en) 2014-03-04 2017-08-01 Bank Of America Corporation Providing offers associated with payment credentials authenticated in a specific digital wallet
US9729536B2 (en) 2015-10-30 2017-08-08 Bank Of America Corporation Tiered identification federated authentication network system
US10511692B2 (en) 2017-06-22 2019-12-17 Bank Of America Corporation Data transmission to a networked resource based on contextual information
US10524165B2 (en) 2017-06-22 2019-12-31 Bank Of America Corporation Dynamic utilization of alternative resources based on token association
US10313480B2 (en) 2017-06-22 2019-06-04 Bank Of America Corporation Data transmission between networked resources
RU2020128190A (ru) * 2018-02-09 2022-03-09 Цзянсу Хэнжуй Медицин Ко., Лтд. Кодон-оптимизированный ген предшественника и ген сигнального пептида аналога человеческого инсулина
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US20120077966A1 (en) 2012-03-29
KR101702342B1 (ko) 2017-02-03
ZA201107067B (en) 2012-05-30
BRPI1008941A2 (pt) 2015-09-01
EP2406377A2 (fr) 2012-01-18
AU2010223154A1 (en) 2011-10-13
US20140057318A1 (en) 2014-02-27
EP2406377A4 (fr) 2012-12-26
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US20140051842A1 (en) 2014-02-20

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